The macrocyclic lactone antibiotic rapamycin is a novel anticancer agent that specifically inhibits the activity of a protein kinase (mTOR) that controls translation of proteins involved in cell cycle progression and survival. Our studies in Project 23 have shown that the response to mTOR inhibition in normal and malignant cells is qualitatively different: rapamycin causes G1 phase arrest and cytostasis in normal cells, whereas tumor cells with deficient G1 checkpoint function (p53/p21) transit G1 phase and undergo apoptosis. Insulin-like growth factor I (IGF-I) uniquely protects against apoptosis. Studies proposed will focus on combining CCI-779, a rapamycin ester undergoing clinical trials, with inhibitors of IGF-I signaling, and further determine the mechanism(s) responsible for acquired and intrinsic resistance to CCI-779. We will attempt to develop biochemical assays to measure mTOR inhibition in tumor tissue, and develop potentially novel combinations of CCI-779 in support our proposed clinical studies (Project 10). Specific hypotheses to be tested are: (1) Inhibition of IGF-I receptor signaling will prevent IGF-I protection against rapamycin-induced apoptosis, and promote apoptosis selectively in cells lacking wild type p53. (2) Regulation of 4E-BP protein levels is a critical determinant of rapamycin sensitivity. (3) Tumor response to high dose CCI-779 is dependent on mTOR inhibition, and is associated with prolonged inhibition of eIF4E-dependent translation. (4) Additive or superadditive activity may be obtained when inhibitors of targets upstream of mTOR or inhibitors of Ras/MAP kinase pathways are combined with rapamycin/CCI-779. Conversely, CCI-779 may synergize or be antagonistic with cytotoxic agents. These studies will extensively use core facilities within this program, and continue to generate leads that may be valuable in clinical testing of CCI-779 alone or in combination with other agents. The longterm goal of these studies is to develop alternative approaches to curative therapy for children with cancer, based on an understanding of IGF-I receptor signaling in growth and survival of pediatric cancer cells.